Lipids and lipid nanoparticle formulations for delivery of nucleic acids

ABSTRACT

Compounds are provided having the following structure: 
                         
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , G 1 , G 2 , G 3 , a, b, c and d are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

BACKGROUND

Technical Field

The present invention generally relates to novel cationic lipids thatcan be used in combination with other lipid components, such as neutrallipids, cholesterol and polymer conjugated lipids, to form lipidnanoparticles with oligonucleotides, to facilitate the intracellulardelivery of therapeutic nucleic acids (e.g. oligonucleotides, messengerRNA) both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto effect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face using oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from cationic lipids withother lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved cationic lipids and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticles would provide optimal drug:lipid ratios, protect thenucleic acid from degradation and clearance in serum, be suitable forsystemic delivery, and provide intracellular delivery of the nucleicacid. In addition, these lipid-nucleic acid particles should bewell-tolerated and provide an adequate therapeutic index, such thatpatient treatment at an effective dose of the nucleic acid is notassociated with unacceptable toxicity and/or risk to the patient. Thepresent invention provides these and related advantages.

BRIEF SUMMARY

In brief, embodiments of the present invention provide lipid compounds,including stereoisomers, pharmaceutically acceptable salts or tautomersthereof, which can be used alone or in combination with other lipidcomponents such as neutral lipids, charged lipids, steroids (includingfor example, all sterols) and/or their analogs, and/or polymerconjugated lipids to form lipid nanoparticles for the delivery oftherapeutic agents. In some instances, the lipid nanoparticles are usedto deliver nucleic acids such as antisense and/or messenger RNA. Methodsfor use of such lipid nanoparticles for treatment of various diseases orconditions, such as those caused by infectious entities and/orinsufficiency of a protein, are also provided.

In one embodiment, compounds having the following Formula (I) areprovided:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), R^(4b),R⁵, R⁶, R⁷, R⁸, R⁹, L¹, L², G¹, G², G³, a, b, c and d are as definedherein.

Pharmaceutical compositions comprising one or more of the foregoingcompounds of Formula (I) and a therapeutic agent are also provided. Insome embodiments, the pharmaceutical compositions further comprise oneor more components selected from neutral lipids, charged lipids,steroids and polymer conjugated lipids. Such compositions are useful forformation of lipid nanoparticles for the delivery of the therapeuticagent.

In other embodiments, the present invention provides a method foradministering a therapeutic agent to a patient in need thereof, themethod comprising preparing a composition of lipid nanoparticlescomprising the compound of Formula (I) and a therapeutic agent anddelivering the composition to the patient.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 shows time course of luciferase expression in mouse liver.

FIG. 2 illustrates the calculation of pKa for MC3 as a representativeexample relevant to the disclosed lipids.

FIG. 3 provides comparative luciferase activity data for differentlipids.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidnanoparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular,embodiments of the present invention provide nucleic acid-lipidnanoparticle compositions comprising one or more of the novel cationiclipids described herein that provide increased activity of the nucleicacid and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid nanoparticle compositions previously described.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of mRNA and/or other oligonucleotides. Insome embodiments, these improved lipid nanoparticle compositions areuseful for expression of protein encoded by mRNA. In other embodiments,these improved lipid nanoparticle compositions are useful forupregulation of endogenous protein expression by delivering miRNAinhibitors targeting one specific miRNA or a group of miRNA regulatingone target mRNA or several mRNA. In other embodiments, these improvedlipid nanoparticle compositions are useful for down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes. Insome other embodiments, the lipid nanoparticles are also useful fordelivery of mRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticle compositions are useful for inducinga pharmacological effect resulting from expression of a protein, e.g.,increased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antibody.

The lipid nanoparticles and compositions of embodiments of the presentinvention may be used for a variety of purposes, including the deliveryof encapsulated or associated (e.g., complexed) therapeutic agents suchas nucleic acids to cells, both in vitro and in vivo. Accordingly,embodiments of the present invention provide methods of treating orpreventing diseases or disorders in a subject in need thereof bycontacting the subject with a lipid nanoparticle that encapsulates or isassociated with a suitable therapeutic agent, wherein the lipidnanoparticle comprises one or more of the novel cationic lipidsdescribed herein.

As described herein, embodiments of the lipid nanoparticles of thepresent invention are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles and compositions of certain embodiments of thepresent invention may be used to induce expression of a desired proteinboth in vitro and in vivo by contacting cells with a lipid nanoparticlecomprising one or more novel cationic lipids described herein, whereinthe lipid nanoparticle encapsulates or is associated with a nucleic acidthat is expressed to produce the desired protein (e.g., a messenger RNAor plasmid encoding the desired protein). Alternatively, the lipidnanoparticles and compositions of some embodiments of the presentinvention may be used to decrease the expression of target genes andproteins both in vitro and in vivo by contacting cells with a lipidnanoparticle comprising one or more novel cationic lipids describedherein, wherein the lipid nanoparticle encapsulates or is associatedwith a nucleic acid that reduces target gene expression (e.g., anantisense oligonucleotide or small interfering RNA (siRNA)). The lipidnanoparticles and compositions of embodiments of the present inventionmay also be used for co-delivery of different nucleic acids (e.g. mRNAand plasmid DNA) separately or in combination, such as may be useful toprovide an effect requiring colocalization of different nucleic acids(e.g. mRNA encoding for a suitable gene modifying enzyme and DNAsegment(s) for incorporation into the host genome).

Nucleic acids for use in embodiments of the invention may be preparedaccording to any available technique. For mRNA, the primary methodologyof preparation is, but is not limited to, enzymatic synthesis (alsotermed in vitro transcription) which currently represents the mostefficient method to produce long sequence-specific mRNA. In vitrotranscription describes a process of template-directed synthesis of RNAmolecules from an engineered DNA template comprised of an upstreambacteriophage promoter sequence (e.g. including but not limited to thatfrom the T7, T3 and SP6 coliphage) linked to a downstream sequenceencoding the gene of interest. Template DNA can be prepared for in vitrotranscription from a number of sources with appropriate techniques whichare well known in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y. Humana Press, 2012)

Transcription of the RNA occurs in vitro using the linearized DNAtemplate in the presence of the corresponding RNA polymerase andadenosine, guanosine, uridine and cytidine ribonucleoside triphosphates(rNTPs) under conditions that support polymerase activity whileminimizing potential degradation of the resultant mRNA transcripts. Invitro transcription can be performed using a variety of commerciallyavailable kits including, but not limited to RiboMax Large Scale RNAProduction System (Promega), MegaScript Transcription kits (LifeTechnologies) as well as with commercially available reagents includingRNA polymerases and rNTPs. The methodology for in vitro transcription ofmRNA is well known in the art. (see, e.g. Losick, R., 1972, In vitrotranscription, Ann Rev Biochem v.41 409-46; Kamakaka, R. T. and Kraus,W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology.2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis ofRNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L.and Green, R., 2013, Chapter Five—In vitro transcription from plasmid orPCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of whichare incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNA oligosetc.). Techniques for the isolation of the mRNA transcripts are wellknown in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (ethanol, isopropanol)in the presence of monovalent cations or lithium chloride. Additional,non-limiting examples of purification procedures which can be usedinclude size exclusion chromatography (Lukaysky, P. J. and Puglisi, J.D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see e.g. Kariko, K., Muramatsu, H., Ludwig,J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLCpurification eliminates immune activation and improves translation ofnucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142;Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLCPurification of in vitro transcribed long RNA in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013). HPLC purified mRNA has been reported tobe translated at much greater levels, particularly in primary cells andin vivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e. cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see e.g. Grudzien-Nogalska, E., Kowalska,J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E., Sahin, U.,Jemielity, J., and Rhoads, R. E., Synthetic mRNAs with superiortranslation and stability properties in Synthetic Messenger RNA and CellMetabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogenous length. 5′-capping and 3′-poly (A) tailingcan be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see e.g., Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v. 10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko,K., In vitro transcription of long RNA containing modified nucleosidesin Synthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v. 969 (Rabinovich, P. H. Ed), 2013); Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v. 16, 1833-1840. The modifiednucleosides and nucleotides used in the synthesis of modified RNAs canbe prepared monitored and utilized using general methods and proceduresknown in the art. A large variety of nucleoside modifications areavailable that may be incorporated alone or in combination with othermodified nucleosides to some extent into the in vitro transcribed mRNA(see e.g., US2012/0251618). In vitro synthesis of nucleoside-modifiedmRNA have been reported to have reduced ability to activate immunesensors with a concomitant enhanced translational capacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see e.g., Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used forembodiments of this invention. For oligonucleotides, methods ofpreparation include but are not limited to chemical synthesis andenzymatic, chemical cleavage of a longer precursor, in vitrotranscription as described above, etc. Methods of synthesizing DNA andRNA nucleotides are widely used and well known in the art (see, e.g.,Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach,Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn,P. (ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

For plasmid DNA, preparation for use with embodiments of this inventioncommonly utilizes, but is not limited to, expansion and isolation of theplasmid DNA in vitro in a liquid culture of bacteria containing theplasmid of interest. The presence of a gene in the plasmid of interestthat encodes resistance to a particular antibiotic (penicillin,kanamycin, etc.) allows those bacteria containing the plasmid ofinterest to selective grow in antibiotic-containing cultures. Methods ofisolating plasmid DNA are widely used and well known in the art (see,e.g. Heilig, J., Elbing, K. L. and Brent, R (2001) Large-ScalePreparation of Plasmid DNA. Current Protocols in Molecular Biology.41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillström, S.,Björnestedt, R. and Schmidt, S. R. (2008), Large-scale production ofendotoxin-free plasmids for transient expression in mammalian cellculture. Biotechnol. Bioeng., 99: 557-566; and US6197553B1). Plasmidisolation can be performed using a variety of commercially availablekits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmidMaxiPrep (Thermo) and PureYield MaxiPrep (Promega) kits as well as withcommercially available reagents.

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid nanoparticles and compositions comprising the same, andtheir use to deliver active (e.g., therapeutic agents), such as nucleicacids, to modulate gene and protein expression, are described in furtherdetail below.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g., asample of cells in culture expressing the desired protein) or a testmammal (e.g., a mammal such as a human or an animal model such as arodent (e.g., mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid (e.g., nucleic acid in combination with alipid of the present invention). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g., a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal model such as a rodent (e.g., mouse) ornon-human primate (e.g., monkey) model) that is not contacted with oradministered the nucleic acid. When the desired protein is present in acontrol sample or a control mammal, the expression of a desired proteinin a control sample or a control mammal may be assigned a value of 1.0.In particular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0, 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g., a sample of cells in culture expressing the target gene) or atest mammal (e.g., a mammal such as a human or an animal model such as arodent (e.g., mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g., a sample of cells in culture expressing the targetgene) or a control mammal (e.g., a mammal such as a human or an animalmodel such as a rodent (e.g., mouse) or non-human primate (e.g., monkey)model) that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g., an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000 or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term “nucleic acid” encompasses nucleic acidscontaining known analogues of natural nucleotides that have similarbinding properties as the reference nucleic acid. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, single nucleotidepolymorphisms, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991);Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes, 8:91-98 (1994)). “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and naturalanalogs, and synthetic derivatives of purines and pyrimidines, whichinclude, but are not limited to, modifications which place new reactivegroups such as, but not limited to, amines, alcohols, thiols,carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. Lipids are usually divided into at least threeclasses: (1) “simple lipids,” which include fats and oils as well aswaxes; (2) “compound lipids,” which include phospholipids andglycolipids; and (3) “derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bear the positive charge. Exemplary cationic lipids are ionizablesuch that they can exist in a positively charged or neutral formdepending on pH. The ionization of the cationic lipid affects thesurface charge of the lipid nanoparticle under different pH conditions.This charge state can influence plasma protein absorption, bloodclearance and tissue distribution (Semple, S. C., et al., Adv. DrugDeliv Rev 32:3-17 (1998)) as well as the ability to form endosomolyticnon-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196(2001)) critical to the intracellular delivery of nucleic acids.

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of formula (I) or other specified cationiclipids. In some embodiments, lipid nanoparticles are included in aformulation that can be used to deliver an active agent or therapeuticagent, such as a nucleic acid (e.g., mRNA) to a target site of interest(e.g., cell, tissue, organ, tumor, and the like). In some embodiments,the lipid nanoparticles of the invention comprise a nucleic acid. Suchlipid nanoparticles typically comprise a compound of Formula (I) and oneor more excipient selected from neutral lipids, charged lipids, steroidsand polymer conjugated lipids. In some embodiments, the active agent ortherapeutic agent, such as a nucleic acid, may be encapsulated in thelipid portion of the lipid nanoparticle or an aqueous space enveloped bysome or all of the lipid portion of the lipid nanoparticle, therebyprotecting it from enzymatic degradation or other undesirable effectsinduced by the mechanisms of the host organism or cells e.g. an adverseimmune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, nucleic acids,when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipid nanoparticles comprisingnucleic acids and their method of preparation are disclosed in, e.g.,U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub.Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of whichare herein incorporated by reference in their entirety for all purposes.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range e.g. pH ˜3 topH ˜9. Charged lipids may be synthetic or naturally derived. Examples ofcharged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates,dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g. DC-Chol).

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intraarterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous or intradermal injection.Local delivery does not preclude a systemic pharmacological effect.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having, for example, from one to twenty-four carbon atoms (C₁-C₂₄alkyl), four to twenty carbon atoms (C₄-C₂₀ alkyl), six to sixteencarbon atoms (C₆-C₁₆ alkyl), six to nine carbon atoms (C₆-C₉ alkyl), oneto fifteen carbon atoms (C₁-C₁₅ alkyl), one to twelve carbon atoms(C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₈ alkyl) or one to sixcarbon atoms (C₁-C₆ alkyl) and which is attached to the rest of themolecule by a single bond, e.g., methyl, ethyl, n propyl, 1 methylethyl(iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but 1 enyl, pent 1enyl, penta 1,4 dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl,and the like. Unless stated otherwise specifically in the specification,an alkyl group is optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving, for example, from one to twenty-four carbon atoms (C₁-C₂₄alkylene), one to fifteen carbon atoms (C₁-C₁₅ alkylene), one to twelvecarbon atoms (C₁-C₁₂ alkylene), one to eight carbon atoms (C₁-C₈alkylene), one to six carbon atoms (C₁-C₆ alkylene), two to four carbonatoms (C₂-C₄ alkylene), one to two carbon atoms (C₁-C₂ alkylene), e.g.,methylene, ethylene, propylene, n-butylene, ethenylene, propenylene,n-butenylene, propynylene, n-butynylene, and the like. The alkylenechain is attached to the rest of the molecule through a single or doublebond and to the radical group through a single or double bond. Thepoints of attachment of the alkylene chain to the rest of the moleculeand to the radical group can be through one carbon or any two carbonswithin the chain. Unless stated otherwise specifically in thespecification, an alkylene chain may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered (e.g., 5, 6 or 7-membered) non-aromatic ring radical havingone to twelve ring carbon atoms (e.g., two to twelve) and from one tosix ring heteroatoms selected from the group consisting of nitrogen,oxygen and sulfur. Unless stated otherwise specifically in thespecification, the heterocyclyl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems; and the nitrogen, carbon or sulfur atoms in theheterocyclyl radical may be optionally oxidized; the nitrogen atom maybe optionally quaternized; and the heterocyclyl radical may be partiallyor fully saturated. Examples of such heterocyclyl radicals include, butare not limited to, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl,tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless statedotherwise specifically in the specification, a heterocyclyl group may beoptionally substituted.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene or heterocyclyl) wherein at least one hydrogen atom(e.g., 1, 2, 3 or all hydrogen atoms) is replaced by a bond to anon-hydrogen atom such as, but not limited to: a halogen atom such as F,Cl, Br, or I; oxo groups (═O); hydroxyl groups (—OH); C₁-C₁₂ alkylgroups; cycloalkyl groups; —(C═O)OR′; —O(C═O)R′; —C(═O)R⁴⁰; ˜OR′;—S(O)_(x)R′; —S—SR′; —C(═O)SR′; —SC(═O)R′; —NR′R′; ˜NR′C(═O)R′;—C(═O)R′; —C(═O)NR′R′; —NR′ C(═O)NR′R′; —OC(═O)NR′R′; —NR′C(═O)OR′;˜NR′S(O)_(x)NR′R′; ˜NR′S(O)_(x)R′; and ˜S(O)_(x)NR′R′, wherein: R′ is,at each occurrence, independently H, C₁-C₁₅ alkyl or cycloalkyl, and xis 0, 1 or 2. In some embodiments the substituent is a C₁-C₁₂ alkylgroup. In other embodiments, the substituent is a cycloalkyl group. Inother embodiments, the substituent is a halo group, such as fluoro. Inother embodiments, the substituent is a oxo group. In other embodiments,the substituent is a hydroxyl group. In other embodiments, thesubstituent is an alkoxy group. In other embodiments, the substituent isa carboxyl group. In other embodiments, the substituent is an aminegroup.

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention (e.g., compound of formula (I)) may beprepared by modifying functional groups present in the compound of theinvention in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound of theinvention. Prodrugs include compounds of the invention wherein ahydroxy, amino or mercapto group is bonded to any group that, when theprodrug of the compound of the invention is administered to a mammaliansubject, cleaves to form a free hydroxy, free amino or free mercaptogroup, respectively. Examples of prodrugs include, but are not limitedto, acetate, formate and benzoate derivatives of alcohol or amidederivatives of amine functional groups in the compounds of the inventionand the like.

Embodiments of the invention disclosed herein are also meant toencompass all pharmaceutically acceptable compounds of the compound ofFormula (I) being isotopically-labelled by having one or more atomsreplaced by an atom having a different atomic mass or mass number.Examples of isotopes that can be incorporated into the disclosedcompounds include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, chlorine, and iodine, such as ²H, 3H, ¹¹C, ¹³C,¹⁴C, ¹⁵N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I,respectively. These radiolabeled compounds can be useful to helpdetermine or measure the effectiveness of the compounds, bycharacterizing, for example, the site or mode of action, or bindingaffinity to pharmacologically important site of action. Certainisotopically-labelled compounds having a structure of Formula (I) or(II), for example, those incorporating a radioactive isotope, are usefulin drug and/or substrate tissue distribution studies. The radioactiveisotopes tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, are particularlyuseful for this purpose in view of their ease of incorporation and readymeans of detection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof Formula (I) of (II) can generally be prepared by conventionaltechniques known to those skilled in the art or by processes analogousto those described in the Preparations and Examples as set out belowusing an appropriate isotopically-labeled reagent in place of thenon-labeled reagent previously employed.

Embodiments of the invention disclosed herein are also meant toencompass the in vivo metabolic products of the disclosed compounds.Such products may result from, for example, the oxidation, reduction,hydrolysis, amidation, esterification, and the like of the administeredcompound, primarily due to enzymatic processes. Accordingly, embodimentsof the invention include compounds produced by a process comprisingadministering a compound of this invention to a mammal for a period oftime sufficient to yield a metabolic product thereof. Such products aretypically identified by administering a radiolabelled compound of theinvention in a detectable dose to an animal, such as rat, mouse, guineapig, monkey, or to human, allowing sufficient time for metabolism tooccur, and isolating its conversion products from the urine, blood orother biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the invention may be truesolvates, while in other cases, the compound of the invention may merelyretain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the invention, or a lipid nanoparticlecomprising the same, which, when administered to a mammal, preferably ahuman, is sufficient to effect treatment in the mammal, preferably ahuman. The amount of a lipid nanoparticle of the invention whichconstitutes a “therapeutically effective amount” will vary depending onthe compound, the condition and its severity, the manner ofadministration, and the age of the mammal to be treated, but can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds of the invention, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. Embodiments of the present invention aremeant to include all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. Embodiments of the present inventioncontemplates various stereoisomers and mixtures thereof and includes“enantiomers”, which refers to two stereoisomers whose molecules arenonsuperimposeable mirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. Embodiments of the present inventioninclude tautomers of any said compounds.

Compounds

In an aspect, the invention provides novel lipid compounds which arecapable of combining with other lipid components such as neutral lipids,charged lipids, steroids and/or polymer conjugated-lipids to form lipidnanoparticles with oligonucleotides. Without wishing to be bound bytheory, it is thought that these lipid nanoparticles shieldoligonucleotides from degradation in the serum and provide for effectivedelivery of oligonucleotides to cells in vitro and in vivo.

In one embodiment, the lipid compounds have the structure of Formula(I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

L¹ and L² are each independently —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,—S(O)_(x)—, —S—S—, —C(═O)S—, —SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,—NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)—, —NR^(a)C(═O)O— or a direct bond;

G¹ is C₁-C₂ alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR^(a)C(═O)— or adirect bond;

G² is —C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NR^(a)— or a direct bond;

G³ is C₁-C₆ alkylene;

R^(a) is H or C₁-C₁₂ alkyl;

R^(1a) and R^(1b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(1b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(2a) and R^(2b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(2b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(3a) and R^(3b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(3b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(4a) and R^(4b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(4b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R⁵ and R⁶ are each independently H or methyl;

R⁷ is C₄-C₂₀ alkyl;

R⁸ and R⁹ are each independently C₁-C₁₂ alkyl; or R⁸ and R⁹, togetherwith the nitrogen atom to which they are attached, form a 5, 6 or7-membered heterocyclic ring;

a, b, c and d are each independently an integer from 1 to 24; and

x is 0, 1 or 2.

In some embodiments, L¹ and L² are each independently —O(C═O)—, —(C═O)O—or a direct bond. In other embodiments, G¹ and G² are each independently—(C═O)— or a direct bond. In some different embodiments, L¹ and L² areeach independently —O(C═O)—, —(C═O)O— or a direct bond; and G¹ and G²are each independently —(C═O)— or a direct bond.

In some different embodiments, L¹ and L² are each independently —C(═O)—,—O—, —S(O)_(x)—, —S—S—, —C(═O)S—, —SC(═O)—, —NR^(a)—, —NR^(a)C(═O)—,—C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a), —OC(═O)NR^(a)—, —NR^(a)C(═O)O—,—NR^(a)S(O)_(x)NR^(a)—, —NR^(a)S(O)_(x)— or —S(O)_(x)NR^(a)—.

In other of the foregoing embodiments, the compound has one of thefollowing structures (IA) or (IB):

In some embodiments, the compound has structure (IA). In otherembodiments, the compound has structure (IB).

In any of the foregoing embodiments, one of L¹ or L² is —O(C═O)—. Forexample, in some embodiments each of L¹ and L² are —O(C═O)—.

In some different embodiments of any of the foregoing, one of L¹ or L²is —(C═O)O—. For example, in some embodiments each of L¹ and L² is—(C═O)O—.

In different embodiments, one of L¹ or L² is a direct bond. As usedherein, a “direct bond” means the group (e.g., L¹ or L²) is absent. Forexample, in some embodiments each of L¹ and L² is a direct bond.

In other different embodiments of the foregoing, for at least oneoccurrence of R^(1a) and R^(1b), R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(1b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In still other different embodiments, for at least one occurrence ofR^(4a) and R^(4b), R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) together withthe carbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

In more embodiments, for at least one occurrence of R^(2a) and R^(2b),R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(2b) and thecarbon atom to which it is bound to form a carbon-carbon double bond.

In other different embodiments of any of the foregoing, for at least oneoccurrence of R^(3a) and R^(3b), R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(3b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

It is understood that “carbon-carbon” double bond refers to one of thefollowing structures:

wherein R^(c) and R^(d) are, at each occurrence, independently H or asubstituent. For example, in some embodiments R^(c) and R^(d) are, ateach occurrence, independently H, C₁-C₁₂ alkyl or cycloalkyl, forexample H or C₁-C₁₂ alkyl.

In various other embodiments, the compound has one of the followingstructures (IC) or (ID):

wherein e, f, g and h are each independently an integer from 1 to 12.

In some embodiments, the compound has structure (IC). In otherembodiments, the compound has structure (ID).

In various embodiments of the compounds of structures (IC) or (ID), e,f, g and h are each independently an integer from 4 to 10.

In certain embodiments of the foregoing, a, b, c and d are eachindependently an integer from 2 to 12 or an integer from 4 to 12. Inother embodiments, a, b, c and d are each independently an integer from8 to 12 or 5 to 9. In some certain embodiments, a is 0. In someembodiments, a is 1. In other embodiments, a is 2. In more embodiments,a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5.In other embodiments, a is 6. In more embodiments, a is 7. In yet otherembodiments, a is 8. In some embodiments, a is 9. In other embodiments,a is 10. In more embodiments, a is 11. In yet other embodiments, a is12. In some embodiments, a is 13. In other embodiments, a is 14. In moreembodiments, a is 15. In yet other embodiments, a is 16.

In some embodiments, b is 1. In other embodiments, b is 2. In moreembodiments, b is 3. In yet other embodiments, b is 4. In someembodiments, b is 5. In other embodiments, b is 6. In more embodiments,b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9.In other embodiments, b is 10. In more embodiments, b is 11. In yetother embodiments, b is 12. In some embodiments, b is 13. In otherembodiments, b is 14. In more embodiments, b is 15. In yet otherembodiments, b is 16.

In some embodiments, c is 1. In other embodiments, c is 2. In moreembodiments, c is 3. In yet other embodiments, c is 4. In someembodiments, c is 5. In other embodiments, c is 6. In more embodiments,c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9.In other embodiments, c is 10. In more embodiments, c is 11. In yetother embodiments, c is 12. In some embodiments, c is 13. In otherembodiments, c is 14. In more embodiments, c is 15. In yet otherembodiments, c is 16.

In some certain embodiments, d is 0. In some embodiments, d is 1. Inother embodiments, d is 2. In more embodiments, d is 3. In yet otherembodiments, d is 4. In some embodiments, d is 5. In other embodiments,d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8.In some embodiments, d is 9. In other embodiments, d is 10. In moreembodiments, d is 11. In yet other embodiments, d is 12. In someembodiments, d is 13. In other embodiments, d is 14. In moreembodiments, d is 15. In yet other embodiments, d is 16.

In some embodiments, e is 1. In other embodiments, e is 2. In moreembodiments, e is 3. In yet other embodiments, e is 4. In someembodiments, e is 5. In other embodiments, e is 6. In more embodiments,e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9.In other embodiments, e is 10. In more embodiments, e is 11. In yetother embodiments, e is 12.

In some embodiments, f is 1. In other embodiments, f is 2. In moreembodiments, f is 3. In yet other embodiments, f is 4. In someembodiments, f is 5. In other embodiments, f is 6. In more embodiments,f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9.In other embodiments, f is 10. In more embodiments, f is 11. In yetother embodiments, f is 12.

In some embodiments, g is 1. In other embodiments, g is 2. In moreembodiments, g is 3. In yet other embodiments, g is 4. In someembodiments, g is 5. In other embodiments, g is 6. In more embodiments,g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9.In other embodiments, g is 10. In more embodiments, g is 11. In yetother embodiments, g is 12.

In some embodiments, h is 1. In other embodiments, e is 2. In moreembodiments, h is 3. In yet other embodiments, h is 4. In someembodiments, e is 5. In other embodiments, h is 6. In more embodiments,h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9.In other embodiments, h is 10. In more embodiments, h is 11. In yetother embodiments, h is 12.

In some other various embodiments, a and d are the same. In some otherembodiments, b and c are the same. In some other specific embodimentsand a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d are factors which may bevaried to obtain a lipid having the desired properties. In oneembodiment, a and b are chosen such that their sum is an integer rangingfrom 14 to 24. In other embodiments, c and d are chosen such that theirsum is an integer ranging from 14 to 24. In further embodiment, the sumof a and b and the sum of c and d are the same. For example, in someembodiments the sum of a and b and the sum of c and d are both the sameinteger which may range from 14 to 24. In still more embodiments, a, b,c and d are selected such that the sum of a and b and the sum of c and dis 12 or greater.

The substituents at R^(1a), R^(2a), R^(3a) and R^(4a) are notparticularly limited. In some embodiments, at least one of R^(1a),R^(2a), R^(3a) and R^(4a) is H. In certain embodiments R^(1a), R^(2a),R^(3a) and R^(4a) are H at each occurrence. In certain other embodimentsat least one of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₁₂ alkyl. Incertain other embodiments at least one of R^(1a), R^(2a), R^(3a) andR^(4a) is C₁-C₈ alkyl. In certain other embodiments at least one ofR^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₆ alkyl. In some of theforegoing embodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of the foregoing, R^(1a), R^(1b), R^(4a) andR^(4b) are C₁-C₁₂ alkyl at each occurrence.

In further embodiments of the foregoing, at least one of R^(1b), R^(2b),R^(3b) and R^(4b) is H or R^(1b), R^(2b), R^(3b) and R^(4b) are H ateach occurrence.

In certain embodiments of the foregoing, R^(1b) together with the carbonatom to which it is bound is taken together with an adjacent R^(1b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond. In other embodiments of the foregoing R^(4b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

The substituents at R⁵ and R⁶ are not particularly limited in theforegoing embodiments. In certain embodiments one of R⁵ or R⁶ is methyl.In other embodiments each of R⁵ or R⁶ is methyl.

The substituents at R⁷ are not particularly limited in the foregoingembodiments. In certain embodiments R⁷ is C₆-C₁₆ alkyl. In some otherembodiments, R⁷ is C₆-C₉ alkyl. In some of these embodiments, R⁷ issubstituted with —(C═O)OR^(b), —O(C═O)R^(b), —C(═O)R^(b), —OR^(b),—S(O)_(x)R^(b), —S—SR^(b), —C(═O)SR^(b), —SC(═O)R^(b), —NR^(a)R^(b),—NR^(a)C(═O)R^(b), —C(═O)NR^(a)R^(b), —NR^(a)C(═O)NR^(a)R^(b),—OC(═O)NR^(a)R^(b), —NR^(a)C(═O)OR^(b), —NR^(a)S(O)_(x)NR^(a)R^(b),—NR^(a)S(O)_(x)R^(b) or —S(O)_(x)NR^(a)R^(b), wherein: R^(a) is H orC₁-C₁₂ alkyl; R^(b) is C₁-C₁₅ alkyl; and x is 0, 1 or 2. For example, insome embodiments R⁷ is substituted with —(C═O)OR^(b) or —O(C═O)R^(b).

In various of the foregoing embodiments, R^(b) is branched C₁-C₁₅ alkyl.For example, in some embodiments R^(b) has one of the followingstructures:

In certain other of the foregoing embodiments, one of R⁸ or R⁹ ismethyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments, R⁸ and R⁹, together with the nitrogenatom to which they are attached, form a 5, 6 or 7-membered heterocyclicring. In some embodiments of the foregoing, R⁸ and R⁹, together with thenitrogen atom to which they are attached, form a 5-membered heterocyclicring, for example a pyrrolidinyl ring. In some different embodiments ofthe foregoing, R⁸ and R⁹, together with the nitrogen atom to which theyare attached, form a 6-membered heterocyclic ring, for example apiperazinyl ring.

In still other embodiments of the foregoing compounds, G³ is C₂-C₄alkylene, for example C₃ alkylene.

In various different embodiments, the compound has one of the structuresset forth in Table 1 below.

TABLE 1 Representative Compounds Preparation No. Structure Method  1

A  2

A  3

A  4

B  5

A  6

A  7

A  8

A  9

A 10

A 11

A 12

A 13

A 14

A 15

A 16

B 17

C 18

A 19

A 20

A 21

A 22

A 23

A 24

A 25

B 26

B 27

B 28

B 29

B 30

B 31

B 32

B 33

B 34

B 35

A 36

C 37

A 38

A 39

A 40

A 41

A 42

A 43

C 44

A 45

A 46

A

It is understood that any embodiment of the compounds of Formula (I), asset forth above, and any specific substituent and/or variable in thecompound Formula (I), as set forth above, may be independently combinedwith other embodiments and/or substituents and/or variables of compoundsof Formula (I) to form embodiments of the inventions not specificallyset forth above. In addition, in the event that a list of substituentsand/or variables is listed for any particular R group, L group, G group,or variables a-h, or x in a particular embodiment and/or claim, it isunderstood that each individual substituent and/or variable may bedeleted from the particular embodiment and/or claim and that theremaining list of substituents and/or variables will be considered to bewithin the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

In some embodiments, compositions comprising any one or more of thecompounds of Formula (I) and a therapeutic agent are provided. Forexample, in some embodiments, the compositions comprise any of thecompounds of Formula (I) and a therapeutic agent and one or moreexcipient selected from neutral lipids, steroids and polymer conjugatedlipids. Other pharmaceutically acceptable excipients and/or carriers arealso included in various embodiments of the compositions.

In some embodiments, the neutral lipid is selected from DSPC, DPPC,DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid isDSPC. In various embodiments, the molar ratio of the compound to theneutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the compositions further comprise a steroid orsteroid analogue. In certain embodiments, the steroid or steroidanalogue is cholesterol. In some of these embodiments, the molar ratioof the compound to cholesterol ranges from about 2:1 to 1:1.

In various embodiments, the polymer conjugated lipid is a pegylatedlipid. For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the compound to the pegylatedlipid ranges from about 100:1 to about 25:1.

In some embodiments, the composition comprises a pegylated lipid havingthe following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R¹⁰ and R¹¹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

z has a mean value ranging from 30 to 60.

In some embodiments, R¹⁰ and R¹¹ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average z is about 45.

In some embodiments of the foregoing composition, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense, plasmid DNA and messenger RNA.

In other different embodiments, the invention is directed to a methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing any of the foregoingcompositions and administering the composition to the patient

For the purposes of administration, the compounds of the presentinvention (typically in the form of lipid nanoparticles in combinationwith a therapeutic agent) may be administered as a raw chemical or maybe formulated as pharmaceutical compositions. Pharmaceuticalcompositions of the present invention comprise a compound of Formula (I)and one or more pharmaceutically acceptable carrier, diluent orexcipient. The compound of Formula (I) is present in the composition inan amount which is effective to form a lipid nanoparticle and deliverthe therapeutic agent, e.g., for treating a particular disease orcondition of interest. Appropriate concentrations and dosages can bereadily determined by one skilled in the art.

Administration of the compositions of the invention can be carried outvia any of the accepted modes of administration of agents for servingsimilar utilities. The pharmaceutical compositions of the invention maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suspensions, suppositories, injections, inhalants, gels,microspheres, and aerosols. Typical routes of administering suchpharmaceutical compositions include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intradermal,intrasternal injection or infusion techniques. Pharmaceuticalcompositions of the invention are formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa subject or patient take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of acompound of the invention in aerosol form may hold a plurality of dosageunits. Actual methods of preparing such dosage forms are known, or willbe apparent, to those skilled in this art; for example, see Remington:The Science and Practice of Pharmacy, 20th Edition (Philadelphia Collegeof Pharmacy and Science, 2000). The composition to be administered will,in any event, contain a therapeutically effective amount of a compoundof the invention, or a pharmaceutically acceptable salt thereof, fortreatment of a disease or condition of interest in accordance with theteachings of this invention.

A pharmaceutical composition of the invention may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose; agents to act as cryoprotectants such assucrose or trehalose. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for eitherparenteral or oral administration should contain an amount of a compoundof the invention such that a suitable dosage will be obtained.

The pharmaceutical composition of the invention may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

The pharmaceutical composition of the invention may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of the invention may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the invention in solid or liquid formmay include an agent that binds to the compound of the invention andthereby assists in the delivery of the compound. Suitable agents thatmay act in this capacity include a monoclonal or polyclonal antibody, ora protein.

The pharmaceutical composition of the invention may consist of dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of the invention may bedelivered in single phase, bi-phasic, or tri-phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols.

The pharmaceutical compositions of the invention may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining the lipid nanoparticles of the invention withsterile, distilled water or other carrier so as to form a solution. Asurfactant may be added to facilitate the formation of a homogeneoussolution or suspension. Surfactants are compounds that non-covalentlyinteract with the compound of the invention so as to facilitatedissolution or homogeneous suspension of the compound in the aqueousdelivery system.

The compositions of the invention, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific therapeutic agent employed; the metabolic stability andlength of action of the therapeutic agent; the age, body weight, generalhealth, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy.

Compositions of the invention may also be administered simultaneouslywith, prior to, or after administration of one or more other therapeuticagents. Such combination therapy includes administration of a singlepharmaceutical dosage formulation of a composition of the invention andone or more additional active agents, as well as administration of thecomposition of the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, a composition of theinvention and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof the invention and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of theinvention can be converted to their free base or acid form by standardtechniques.

The following Reaction Scheme illustrates methods to make compounds ofthis invention, i.e., compounds of formula (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), R^(4b),R⁵, R⁶, R⁷, R⁸, R⁹, L¹, L², G¹, G², G³, a, b, c and d as defined herein.It is understood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othercompounds of Formula (I) not specifically illustrated below by using theappropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

Embodiments of the compound of structure (I) (e.g., compounds A-5 andA-7) can be prepared according to General Reaction Scheme 1 (“MethodA”), wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a),R^(4b), R⁵, R⁶, R⁸, R⁹, L¹, L², G¹, G², G³, a, b, c and d are as definedherein, and R^(7′) represents R⁷ or a C₃-C₁₉ alkyl. Referring to GeneralReaction Scheme 1, compounds of structure A-1 and A2 can be purchasedfrom commercial sources or prepared according to methods familiar to oneof ordinary skill in the art. A solution of A-1 and A-2 is treated witha reducing agent (e.g., sodium triacetoxyborohydride) to obtain A-3after any necessary work up. A solution of A-3 and a base (e.g.trimethylamine, DMAP) is treated with acyl chloride A-4 (or carboxylicacid and DCC) to obtain A-5 after any necessary work up and/orpurification. A-5 can be reduced with LiAlH4 A-6 to give A-7 after anynecessary work up and/or purification.

Embodiments of the compound of structure (I) (e.g., compound B-5) can beprepared according to General Reaction Scheme 2 (“Method B”), whereinR^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), R^(4b), R⁵, R⁶,R⁷, R⁸, R⁹, L¹, L², G³, a, b, c and d are as defined herein. Referringto General Reaction Scheme 2, compounds of structure B-1 and B-2 can bepurchased from commercial sources or prepared according to methodsfamiliar to one of ordinary skill in the art. A mixture of B-1 (inexcess), B-2 and a base (e.g., potassium carbonate) is heated to obtainB-3 after any necessary work up. A solution of B-3 and a base (e.g.trimethylamine, DMAP) is treated with acyl chloride B-4 (or carboxylicacid and DCC) to obtain B-5 after any necessary work up and/orpurification.

Other embodiments of the compound of Formula (I) (e.g., C-9) areprepared according to General Reaction Scheme 3. As illustrated inGeneral Reaction Scheme 3, an appropriately protected ketone (C-1) isreacted under reductive amination conditions with amine C-2 to yieldC-3. Acylation of C-3 with acid chloride C-4 yields acylated productC-5. Removal of the alcohol protecting group on C-5 followed by reactionwith C-7 and/or C-8 and appropriate activating reagent (e.g., DCC)yields the desired compound C-9.

The following examples are provided for purpose of illustration and notlimitation.

Example 1 Synthesis of Compound 1

Compound 1 was prepared according to method A from compound 5 to yield240 mg of colorless oil, 0.32 mmol, 61%). 1HNMR (400 MHz, CDCl3) δ:5.43-5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H), 2.39-2.25 (m, 7H), 2.22 (s,6H), 2.06 (q, 6.8 Hz, 8H), 1.53 (quintet, 7.3 Hz, 2H), 1.41-1.11 (54H),0.92-0.87 (m, 9H).

Example 2 Synthesis of Compound 2

Compound 2 was prepared according to method A as follows:

Compound 7 (0.84 g, 0.96 mmol) was dissolved in THF (15 mL) and LAH (2eq. 1.92 mmol, 73 mg, MW37.95) was added in portions at RT. After thereaction mixture was heated at 60 C overnight, sodium sulfate hydratewas added. The mixture was stirred for 2 h, filtered through a layer ofsilica gel. The filtrate was concentrated to give a slightly yellow oil(0.86 g). The crude product was purified by gravity columnchromatography on silica gel (0 to 4% MeOH in chloroform). This gave thedesired product as a colorless oil (420 mg, 0.49 mmol, 51%). 1HNMR (400MHz, CDCl3) δ: 5.43-5.30 (m, 12H), 2.78 (t, 6.4 Hz, 6H), 2.40-2.25 (m,7H), 2.22 (s, 6H), 2.06 (q, 6.8 Hz, 12H), 1.53 (quintet, 7.3 Hz, 2H),1.41-1.10 (58H), 0.90 (t, 6.8 Hz, 9H).

Example 3 Synthesis of Compound 3

Compound 3 was prepared according to method A from compound 8 to yield123 mg of colorless oil, 0.15 mmol, 41%). 1HNMR (400 MHz, CDCl3) δ:5.43-5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H), 2.35-2.24 (m, 5H), 2.22 (s,6H), 2.15 (d, 5.5 Hz, 2H), 2.06 (q, 6.8 Hz, 8H), 1.52 (quintet, 7.3 Hz,2H), 1.40-1.09 (65H), 0.92-0.87 (m, 12H).

Example 4 Synthesis of Compound 5

Compound 5 was prepared according to method A as follows:

Step 1

3-dimethylamine-1-propylamine (6 mmol, 612 mg) and the ketone 5a (3.16g, 6 mmol) were mixed in DCE (25 mL) and then treated with sodiumtriacetoxyborohydride (8.49 mmol, 1.8 g) and AcOH (6 mmol, 0.36 g, 0.340mL). The mixture was stirred at rt under a Ar atmosphere for 2 days. Thereaction mixture was quenched by adding 1 N NaOH (ca 20 mL), and theproduct was extracted with a mixture of hexane and ethyl acetate (ca5%). The organic extract was washed with water/brine (1:1), brine anddried (Na2SO4). Concentrated to give the desired product 5b as a yellowoil (3.55 g). The crude product was used for the next step without anyfurther purification.

Step 2

A solution of nonanoyl chloride (212 mg, 1.2 mmol) in benzene (10 mL)was added via syringe to a solution of compound 5b (600 mg, 0.978 mmol)and triethylamine (5 mmol, 0.7 mL, 5 eq) and DMAP (20 mg) in benzene (10mL) at RT in 10 min. After addition, the mixture was then diluted with amixture of hexane and ethyl acetate (ca 5%), washed with water, washedwith brine, dried over sodium sulfate, filtered and concentrated. Thecrude product (0.77 g) was purified by gravity column chromatography onsilica gel (230-400 mesh silica gel, 40 g, MeOH in chloroform, 0 to 4%).This gave the desired product 5 as a colorless oil (563 mg, 0.75 mmol,76%). 1HNMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 4.56-4.36 (br., 0.3H,due to slow isomerization about amide bond), 3.64 (quintet, 7 Hz, 0.7H),3.12-3.09 (m, 2H), 2.78 (t, 6.4 Hz, 4H), 2.33-2.25 (m, 4H), 2.23, 2.22(two sets of singlet, 6H), 2.06 (q-like, 6.8 Hz, 8H), 1.76-1.66 (m, 4H),1.50-1.40 (m, 4H), 1.40-1.15 (46H), 0.90 (t, 6.7 Hz, 6H), 0.88 (t, 6.8Hz, 3H).

Example 5 Synthesis of Compound 6

Compound 6 was prepared according to the general procedure A to yield0.98 g of slightly yellow oil, 1.13 mmol, 58%. 1HNMR (400 MHz, CDCl3) δ:5.43-5.30 (m, 12H), 4.55-4.32 (br., 0.3H, due to slow isomerizationabout amide bond), 3.63 (quintet-like, 7 Hz, 0.7H), 3.15-3.09 (m, 2H),2.78 (t, 6.4 Hz, 6H), 2.33-2.25 (m, 4H), 2.22, 2.23 (two sets ofsinglet, 6H), 2.06 (q-like, 6.8 Hz, 12H), 1.76-1.60 (m, 4H), 1.49-1.16(54H), 0.90 (t-like, 6.8 Hz, 9H).

Example 6 Synthesis of Compound 7

Compound 7 was prepared according to method A as follows:

To a solution of 2-ethylheptanoic acid (1.5 eq. 0.83 mmol, 130 mg) inbenzene (6 mL) and DMF (5-10 uL) was added oxalyl chloride (5 eq, 2.8mmol, 349 mg, 0.24 mL) at RT. The mixture was stirred at RT for 30 minand then heated at 60 C for 2 h under Ar. The mixture was concentrated.The residue was taken up in benzene (6 mL) and concentrated again toremove any oxalyl chloride. The residual oil (light yellow) was taken in4 mL of benzene and added via syringe to a solution of compound 5b (1eq., 0.55 mmol, 337 mg) and triethylamine (5 eq, 2.8 mmol, 283 mg, 390uL) and DMAP (10 mg) in benzene (6 mL) at RT in 10 min. After addition,the resulting mixture was stirred at RT overnight. TLC showed that therewas not much reaction. The reaction was concentrated and dried well andused in the following. The residue was taken up in DCM (20 mL). DMAP(200 mg, 1.64 mmol) was added, followed by addition of DCC (1.64 mmol,338 mg). The mixture was stirred for 11 days and filtered. The filtratewas washed with 5% NaOH (100 mL). The organic phase was washed withbrine, dried over sodium sulfate. Filtration and Concentration gavelight brown oil (0.89 g). The crude product (0.89 g) was purified bycolumn chromatography on silica gel (0 to 4% MeOH in chloroform). Thisgave the desired product as a colorless oil (122 mg, 0.16 mmol, 29%).¹HNMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 4.69-4.51 (very br.,estimated 0.4H, due to slow isomerization about amide bond), 3.72(quintet-like, 6.9 Hz, 0.6H), 3.19-3.09 (m, 2H), 2.78 (t, 6.4 Hz, 4H),2.55 (quintet-like, 6.5 Hz, 0.5H), 2.42 (quintet-like, 6.5 Hz, 0.5H),2.29 (q-like, but could be two overlap triplets, 6.9 Hz, 2H), 2.24, 2.23(two sets of singlet, integration ratio is about 1:1, 6H), 2.09-2.02 (m,8H), 1.77-1.58 (m, 4H), 1.55-1.15 (48H), 0.93-0.85 (m, 12H).

Example 7 Synthesis of Compound 8

Compound 8 was prepared according to the general procedure A to yield0.39 g of colorless oil, 0.46 mmol, 56%. 1HNMR (400 MHz, CDCl3) δ:5.43-5.30 (m, 8H), 4.55-4.32 (very br., estimated 0.3H, due to slowisomerization about amide bond), 3.71 (quintet-like, 7 Hz, 0.7H),3.17-3.08 (m, 2H), 2.78 (t, 6.4 Hz, 4H), 2.59 (quintet-like, 6.5 Hz,0.5H), 2.46 (quintet-like, 6.5 Hz, 0.5H), 2.40 (t, 7 Hz, 1H), 2.31 (t, 7Hz, 1H), 2.28, 2.25 (two sets of singlet, integration ratio is about1:1, 6H), 2.09-2.02 (m, 8H), 1.79-1.69 (m, 2H), 1.66-1.57 (m, 2H),1.55-1.16 (62H), 0.92-0.86 (m, 12H).

Example 8 Synthesis of Compound 9

Compound 9 was prepared according to method A as follows:

Step 1

3-dimethylamine-1-propylamine (1 eq. 1.3 mmol, 133 mg, 163 uL; MW102.18,d 0.812) and the ketone 9a (1 eq., 0.885 g, 1.3 mmol) were mixed in DCE(8 mL) and then treated with sodium triacetoxyborohydride (1.4 eq., 1.82mmol, 386 mg; MW211.94) and AcOH (1 eq., 1.3 mmol, 78 mg, 74 uL, MW60.05, d 1.06). The mixture was stirred at RT under an Ar atmosphere for2 days. The reaction mixture was diluted with hexanes-EtOAc (9:1) andquenched by adding 0.1 N NaOH (20 mL). The organic phase was separated,washed with sat NaHCO3, brine, dried over sodium sulfate, decanted andconcentrated to give the desired product 9b as a slightly yellow cloudyoil (1.07 g, 1.398 mmol).

Step 2

A solution of nonanoyl chloride (1.3 eq., 1.27 mmol, 225 mg) in benzene(10 mL) was added via syringe to a solution of the compound 9b from step1(0.75 g, 0.98 mmol) and triethylamine (5 eq, 4.90 mmol, 0.68 mL) andDMAP (20 mg) in benzene (10 mL) at RT in 10 min. After addition, themixture was stirred at RT overnight. Methanol (5.5 mL) was added toremove excess acyl chloride. After 3 h, the mixture was filtered througha pad of silica gel (1.2 cm). Concentration gave a colorless oil (0.70g).

The crude product (0.70 g) was purified by flash dry columnchromatography on silica gel (0 to 4% MeOH in chloroform). This yielded457 mg of colorless oil, 0.50 mmol, 51%. 1HNMR (400 MHz, CDCl3) δ:4.54-4.36 (very br., estimated 0.3H, due to slow isomerization aboutamide bond), 3.977, 3.973 (two sets of doublets, 5.8 Hz, 4H), 3.63(quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H),2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 10H), 1.49-1.39 (m,4H), 1.37-1.11 (62H), 0.92-0.86 (m, 15H).

Example 9 Synthesis of Compound 10

Compound 10 was prepared according to the general procedure A to yield245 mg of colorless oil, 0.27 mmol, total yield 53% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.87 (quintet-like, 6.3 Hz, 2H), 4.54-4.36 (verybr., estimated 0.3H, due to slow isomerization about amide bond), 3.63(quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H),2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m,12H), 1.37-1.11 (60H), 0.92-0.86 (m, 15H).

Example 10 Synthesis of Compound 11

Compound 11 was prepared according to the general procedure A to yield239 mg of colorless oil, 0.26 mmol, total yield 52% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.87 (quintet-like, 6.3 Hz, 2H), 4.54-4.36 (verybr., estimated 0.3H, due to slow isomerization about amide bond), 3.63(quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H),2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m,12H), 1.37-1.11 (62H), 0.92-0.86 (m, 15H).

Example 11 Synthesis of Compound 12

Compound 12 was prepared according to the general procedure A to yield198 mg of colorless oil, 0.20 mmol, total yield 46% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.54-4.36 (very br., estimated 0.3H, due to slowisomerization about amide bond), 3.974, 3.971 (two sets of doublets, 5.8Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25(m, 8H), 2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 10H),1.49-1.39 (m, 4H), 1.37-1.11 (76H), 0.92-0.86 (m, 15H).

Example 12 Synthesis of Compound 13

Compound 13 was prepared according to the general procedure A to yield217 mg of colorless oil, 0.21 mmol, total yield 49% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.54-4.36 (very br., estimated 0.3H, due to slowisomerization about amide bond), 3.973, 3.970 (two sets of doublets, 5.8Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25(m, 8H), 2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 10H),1.49-1.39 (m, 4H), 1.37-1.11 (78H), 0.92-0.86 (m, 15H).

Example 13 Synthesis of Compound 14

Compound 14 was prepared according to the general procedure A to yield263 mg of colorless oil, 0.29 mmol, total yield 39% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.54-4.36 (br., estimated 0.3H, due to slowisomerization about amide bond), 3.977, 3.973 (two sets of doublets, 5.8Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43(m, 6H), 2.34-2.26 (m, 6H), 1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H),1.49-1.38 (m, 4H), 1.37-1.11 (60H), 0.92-0.86 (m, 15H).

Example 14 Synthesis of Compound 15

Compound 15 was prepared according to the general procedure A to yield234 mg of colorless oil, 0.25 mmol, total yield 34% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.54-4.36 (br., estimated 0.3H, due to slowisomerization about amide bond), 3.977, 3.973 (two sets of doublets, 5.8Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43(m, 6H), 2.34-2.26 (m, 6H), 1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H),1.49-1.38 (m, 4H), 1.37-1.11 (62H), 0.92-0.86 (m, 15H).

Example 15 Synthesis of Compound 16

Compound 16 was prepared according to method B as follows:

To a solution of the acid 018-19 (0.5 g, 0.90 mmol),N-hydroxysuccinimide (1.2 eq, 1.08 mmol, 124 mg) and DMAP (0.3 eq, 0.27mmol, 33 mg) in DCM (20 mL) was added DCC (2 eq, 1.8 mmol, 371 mg). Theresulting mixture was stirred at RT for 16 h. The reaction mixture wasthen filtered and added into a solution of the amine 021-24 (1.26 mmol,288 mg) in DCM (10 mL) and triethylamine (5 mmol, 696 uL). After 15days, the mixture was concentrated. The residue was taken up inhexane/ethyl acetate/Et3N (ca 9:1:0.3) and was filtered through a smallpad of silica gel, washed with a mixture of hexane/ethyl acetate/Et3N(ca 9:1:0.3). The filtrate was concentrated and a yellow oil wasobtained (580 mg). The yellow oil was purified by column chromatographyon silica gel (eluted with a gradient mixture of MeOH in Chloroform, 0to 4.2%). This gave the desired product as a colorless oil (102 mg, 0.13mmol, 14%). ¹HNMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 3.38-3.29 (m,3H), 3.28-3.23 (m, 1H), 2.78 (t, 6.4 Hz, 4H), 2.56-2.47 (m, 1H),2.30-2.24 (m, 2H), 2.23, 2.22 (two sets of singlet, 6H), 2.09-2.02 (m,8H), 1.71 (quintet-like, 7.4 Hz, 2H), 1.66-1.48 (overlapped with water;estimated 4H), 1.47-1.18 (m, 50H), 0.92-0.86 (m, 9H).

Example 16 Synthesis of Compound 24

Compound 24 was prepared according to the general procedure A to yield279 mg of slightly yellow oil, 0.29 mmol, total yield 44% for 2 steps.¹HNMR (400 MHz, CDCl3) δ: 4.88 (quintet-like, 6.3 Hz, 3H), 3.62(quintet-like, 6.8 Hz, 1H), 3.14-3.08 (m, 2H), 2.33-2.25 (m, 10H), 2.23,2.22 (two sets of singlet, 6H), 1.76-1.58 (m, 10H), 1.52 (q-like, 6.7Hz, 12H), 1.49-1.39 (m, 4H), 1.38-1.14 (50H), 0.89 (t-like, 18H).

Example 17 Synthesis of Compound 35

Compound 35 was prepared according to the general procedure A to yield260 mg of slightly yellow oil, 0.29 mmol, total yield 33% for 2 steps.¹HNMR (400 MHz, CDCl3) δ: 4.66-4.52 (very br., estimated 0.3H, due toslow isomerization about amide bond), 3.977, 3.973 (two sets ofdoublets, 5.8 Hz, 4H), 3.71 (quintet-like, 6.8 Hz, 0.7H), 3.19-3.09 (m,2H), 2.54, 2.42 (two sets of quintet-like, 6.8 Hz, integration ratio isabout 1:1.2, 1H), 2.33-2.25 (m, 6H), 2.24, 2.22 (two sets of singlet,6H), 1.77-1.11 (74H), 0.93-0.85 (m, 18H).

Example 18 Synthesis of Compound 17

Compound 17 was prepared according to method C as follows:

Step 1

3-dimethylamino-1-propylamine (1 eq. 4.14 mmol, 423 mg, 521 uL) andketone 17a (1 eq., 2.0 g, 4.14 mmol) were mixed in DCE (30 mL) and thentreated with sodium triacetoxyborohydride (1.4 eq., 5.80 mmol, 1.229 g)and AcOH (1 eq., 4.14 mmol, 249 mg, 235 uL). The mixture was stirred atRT under Ar atmosphere for 2 days.

The reaction mixture was diluted with a mixture of hexanes and EtOAc(9:1, 200 mL) and quenched by adding dilute NaOH solution (0.1 N, 270mL). The two phases were separated. The organic phase was washed withsat NaHCO3, brine, dried over sodium sulfate and filtered through a padof silica gel. The pad was washed with 200 mL of a mixture of hexane andEtOAc (9:1). Then the pad was washed 200 mL of a mixture ofDCM/MeOH/Et3N (85:15:1). The DCM/MeOH/Et3N washing was concentrated togive the desired product (17b) as a colorless oil (1.749 g, 3.07 mmol,74%).

Step 2

A solution of nonanoyl chloride (0.333 mL) in benzene (10 mL) was addedto a solution of compound 17b (0.75 g) and triethylamine (0.92 mL) andDMAP (20 mg) in benzene (20 mL) at RT. The mixture was stirred at RTovernight. MeOH (1 mL) was added and the mixture continued to stir for 2h. The reaction mixture was filtered through a pad of silica gel.Concentration of the filtrate gave the desired product (17c) as a yellowoil (0.945 g).

Step 3

To a flask containing 17c (0.945 g, 1.33 mmol and EtOH (25 mL) was addedp-toluenesulfonic acid hydrate (1.33 mmol, 253 mg) at room temperature.The resulting mixture was stirred overnight at RT. The reaction mixturewas heated at 85 C for 2 h. More PTSA (160 mg) was added and thereaction mixture continued to heat at 75 C overnight. The mixture wasconcentrated. The residue was taken up in DCM and washed with diluteNH4OH solution. The organic phase was washed with a mixture of satsodium bicarbonate and brine; dried over sodium sulfate. Concentrationgave the desired product (17d) as a slightly yellow viscous oil (0.799g, 1.47 mmol). The crude product was purified by silica gel columnchromatography (0 to 15% methanol in DCM with trace of triethlyamine).This gave 17d as a colorless oil (647 mg, 1.20 mmol, 90%).

Step 4

To a solution of 17d (216 mg, 0.40 mmol), 2-butyloctanoic acid (5 eq, 2mmol, 401 mg), and 4-dimethylaminopyridine (DMAP) (5.5 eq. 2.2 mmol, 269mg) in dichloromethane (20 mL) was added DCC (5.5 eq, 2.2 mmol, 454 mg).After being stirred over for 4 days, 3 mL of MeOH was added. The mixturecontinued to stir for another 16 h. The mixture was filtered and thefiltrate was concentrated to dryness. The crude product was purified bygravity column chromatography on silica gel (MeOH in DCM, 0 to 6%). Thisgave the desired compound (17) as a slightly yellow oil (colorless oil,175 mg, 0.19 mmol, 48%). ¹HNMR (400 MHz, CDCl3) δ: 4.07, 4.06 (two setsof triplets, 6.7 Hz, 4H), 3.64 (quintet-like, 6.8 Hz, 1H), 3.21-3.09(two sets of multiplets, 2H), 3.00-2.37 (br. 6H), 2.36-2.20 (m, 6H),2.05-1.85 (m, 2H), 1.79-1.53 (m, 10H), 1.52-1.39 (m, 8H), 1.37-1.03(58H), 0.91-0.86 (m, 15H).

Example 19 Synthesis of Compound 36

Compound 36 was prepared according to the general procedure C to yield156 mg of colorless oil, 0.15 mmol, 38% for the last step. ¹HNMR (400MHz, CDCl3) δ: 4.07 (triplets, 6.7 Hz, 4H), 3.65 (quintet-like, 6.8 Hz,1H), 3.21 (t-like, 6.8 Hz, 2H), 3.10-3.03 (br. 2H), 2.79, 2.78 (two setsof singlet, 6H), 2.35-2.28 (m, 4H), 2.09 (quintet-like, 7.5 Hz, 2H),1.67-1.54 (m, 10H), 1.54-1.38 (m, 8H), 1.38-1.03 (74H), 0.91-0.86 (m,15H).

Example 20 Synthesis of Compound 37

Compound 37 was prepared according to the general procedure A to yield397 mg of colorless oil, 0.49 mmol, total yield 60% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 4.13 (q, 7.1 Hz, 2H), 4.56-4.34(br. 0.3H), 3.63 (quintet-like, 6.9 Hz, 0.7H), 3.15-3.08 (m, 2H), 2.78(t-like, 6.4 Hz, 4H), 2.39-2.21 (m, 12H), 2.06 (q-like, 6.9 Hz, 8H),1.79-1.55 (m, 6H), 1.50-1.40 (m, 4H), 1.40-1.15 (m, 45H), 0.90 (t-like,6.8 Hz, 6H).

Example 21 Synthesis of Compound 38

Compound 38 was prepared according to method A as follows:

Step 1

To a solution of 38a (1 eq., 1.266 g, 1.79 mmol) in DCE (15 mL) wasadded 3-dimethylamino-1-propylamine (1 eq. 1.79 mmol, 183 mg, 225 uL),followed by addition of sodium triacetoxyborohydride (1.4 eq., 2.51mmol, 531 mg) and AcOH (1 eq., 1.79 mmol, 107 mg, 101 uL). The mixturewas stirred at RT under Ar atmosphere for 3 days.

The residue was diluted with hexanes-EtOAc (9:1, 150 mL) and washed withdilute NaOH solution (0.12 N, 100 mL), sat NaHCO3, brine and dired oversodium sulfate. The organic phase was filtered through a pad of silicagel. The pad was washed with 200 mL of a mixture of hexane and EtOAc(9:1). Then the pad was washed with 200 mL of a mixture of DCM/MeOH/Et3N(85:15:1). The DCM/MeOH/Et3N washing was concentrated and dried on highvacuum line to give the desired product (38b) as a colorless oil (1.1 g,1.38 mmol, 77%).

Step 2

A solution of nonanoyl chloride (1.5 eq., 0.68 mmol, 120 mg) in benzene(5 mL) was added to a solution of 38b (0.45 mmol, 360 mg) andtriethylamine (5 eq, 2.25 mmol, 228 mg, 314 uL) and DMAP (10 mg) inbenzene (10 mL) at RT in 2 min under Ar. After addition, the mixture wasstirred at RT overnight. MeOH (1 mL) was added and the mixture continuedto stir 2 h. The crude was filtered through a pad of silica gel. Thefiltrate was concentrated. The residue (457 mg) was purified by flashcolumn chromatography on silica gel (230-400 mesh silica gel, 40 g, MeOHin chloroform, 0 to 4.6%). This gave the desired product (38) as acolorless oil (410 mg, 0.44 mmol, 98%). ¹HNMR (400 MHz, CDCl3) δ:4.61-4.35 (br., estimated 0.4H, due to slow isomerization about amidebond), 3.974, 3.964 (two sets of doublets, 5.7 Hz, 4H), 3.64(quintet-like, 7.0 Hz, 0.6H), 3.14-3.08 (m, 2H), 2.34-2.25 (m, 8H), 2.23(broad s, 6H), 1.77-1.58 (m, 10H), 1.53-1.39 (m, 4H), 1.37-1.15 (66H),0.92-0.86 (m, 15H).

Example 22 Synthesis of Compound 39

Compound 39 was prepared according to the general procedure A to yield370 mg of colorless oil, 0.40 mmol, total yield 69% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.61-4.35 (br., estimated 0.4H, due to slowisomerization about amide bond), 3.974, 3.964 (two sets of doublets, 5.7Hz, 4H), 3.64 (quintet-like, 7.0 Hz, 0.6H), 3.14-3.08 (m, 2H), 2.34-2.25(m, 8H), 2.230, 2.221 (two sets of singlet, 6H), 1.75-1.58 (m, 10H),1.51-1.39 (m, 4H), 1.37-1.15 (64H), 0.92-0.86 (m, 15H).

Example 23 Synthesis of Compound 40

Compound 40 was prepared according to the general procedure A to yield382 mg of colorless oil, 0.39 mmol, total yield 68% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.60-4.35 (br., estimated 0.3H, due to slowisomerization about amide bond), 4.13 (q, 7.2 Hz, 2H), 3.973, 3.964 (twosets of doublets, 5.7 Hz, 4H), 3.63 (quintet-like, 7.0 Hz, 0.7H),3.14-3.08 (m, 2H), 2.34-2.25 (m, 10H), 2.229, 2.220 (two sets ofsinglet, 6H), 1.75-1.58 (m, 12H), 1.51-1.39 (m, 4H), 1.37-1.15 (64H),0.89 (t-like, 7.8 Hz, 12H).

Example 24 Synthesis of Compound 41

Compound 41 was prepared according to the general procedure A to yield309 mg of colorless oil, 0.30 mmol, total yield 73% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.60-4.35 (br., estimated 0.3H, due to slowisomerization about amide bond), 3.972, 3.962 (two sets of doublets, 5.7Hz, 4H), 3.64 (quintet-like, 7.1 Hz, 0.7H), 3.14-3.08 (m, 2H), 2.34-2.25(m, 8H), 2.23, 2.22 (two sets of singlet, 6H), 1.75-1.58 (m, 10H),1.51-1.39 (m, 4H), 1.35-1.21 (82H), 0.92-0.86 (m, 15H).

Example 25 Synthesis of Compound 42

Compound 42 was prepared according to the general procedure A to yield235 mg of colorless oil, 0.23 mmol, total yield 56% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.75-4.49 (br., estimated 0.4H, due to slowisomerization about amide bond), 3.97, 3.96 (two sets of doublets, 5.3Hz, 4H), 3.72 (quintet-like, 7 Hz, 0.6H), 3.21-3.05 (m, 2H), 2.53, 2.42(two sets of quintet-like, 6.6 Hz, integration ratio is about 1:1.7,1H), 2.32-2.25 (m, 6H), 2.24, 2.22 (two sets of singlet, 6H), 1.78-1.56(m, 10H), 1.53-1.39 (m, 6H), 1.38-1.17 (76H), 0.93-0.85 (m, 18H).

Example 26 Synthesis of Compound 43

Compound 43 was prepared according to the general procedure C to yield187 mg of colorless oil, 0.23 mmol, 57% for the last step. ¹HNMR (400MHz, CDCl3) δ: 4.077, 4.071 (two sets of triplets, 6.7 Hz, 4H),4.56-4.34 (br. 0.3H), 3.64 (quintet-like, 6.9 Hz, 0.7H), 3.15-3.09 (m,2H), 2.34-2.24 (m, 6H), 2.234-2.224 (two sets of singlet, 6H), 1.76-1.58(m, 10H), 1.55-1.39 (m, 8H), 1.39-1.10 (48H), 0.92-0.86 (m, 15H).

Example 27 Synthesis of Compound 44

Compound 44 was prepared according to the general procedure A to yield260 mg of colorless oil, 0.22 mmol, total yield 53% for 2 steps. ¹HNMR(400 MHz, CDCl3) δ: 4.59-4.35 (br., estimated 0.3H, due to slowisomerization about amide bond), 4.03-3.95 (m, 6H), 3.63 (quintet-like,6.9 Hz, 0.7H), 3.14-3.08 (m, 2H), 2.33-2.24 (m, 10H), 2.229, 2.221 (twosets of singlet, 6H), 1.75-1.57 (m, 12H), 1.51-1.40 (m, 4H), 1.40-1.08(87H), 0.92-0.86 (m, 18H).

Example 28 Luciferase mRNA in Vivo Evaluation Using the LipidNanoparticle Compositions

Cationic lipid (MC3), DSPC, cholesterol and PEG-lipid were solubilizedin ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNP)were prepared at a total lipid to mRNA weight ratio of approximately10:1 to 30:1. Briefly, the mRNA was diluted to 0.2 mg/mL in 10 to 50 mMcitrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipidsolution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3(vol/vol) with total flow rates above 15 ml/min. The ethanol was thenremoved and the external buffer replaced with PBS by dialysis. Finally,the lipid nanoparticles were filtered through a 0.2 μm pore sterilefilter. Lipid nanoparticle particle size was 70-90 nm diameter asdetermined by quasi-elastic light scattering using a Nicomp 370submicron particle sizer (Santa Barbara, Calif.).

Studies were performed in 6-8 week old female C57BL/6 mice (CharlesRiver) according to guidelines established by an institutional animalcare committee (ACC) and the Canadian Council on Animal Care (CCAC).Varying doses of mRNA-lipid nanoparticle were systemically administeredby tail vein injection and animals euthanized at specific time points(1, 2, 4, 8 and 24 hrs) post-administration. Liver and spleen werecollected in pre-weighted tubes, weights determined, immediately snapfrozen in liquid nitrogen and stored at −80° C. until processing foranalysis.

For liver, approximately 50 mg was dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon Ohio). ¼″ ceramic sphere (MPBiomedicals) was added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature was added toliver tissue. Liver tissues were homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate wasincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed wasdetermined by using the BCA protein assay kit (Pierce, Rockford Ill.).Relative luminescence units (RLU) were then normalized to total ugprotein assayed. To convert RLU to ng luciferase a standard curve wasgenerated with QuantiLum Recombinant Luciferase (Promega). Based in thedata provided in FIG. 1, the four-hour time point was chosen forefficacy evaluation of the lipid formulations (see Example 29).

The FLuc mRNA (L-6107) from Trilink Biotechnologies will express aluciferase protein, originally isolated from the firefly, Photinuspyralis. FLuc is commonly used in mammalian cell culture to measure bothgene expression and cell viability. It emits bioluminescence in thepresence of the substrate, luciferin. This capped and polyadenylatedmRNA is fully substituted with 5-methylcytidine and pseudouridine.

Example 29 Determination of Efficacy of Lipid Nanoparticle FormulationsContaining Various Cationic Lipids Using an in Vivo Luciferase mRNAExpression Rodent Model

The cationic lipids shown in Table 2 have previously been tested withnucleic acids. For comparative purposes, these lipids were also used toformulate lipid nanoparticles containing the FLuc mRNA (L-6107) using anin line mixing method, as described in example 28 and in PCT/US10/22614,which is hereby incorporated by reference in its entirety. Lipidnanoparticles were formulated using the following molar ratio: 50%Cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38.5%Cholesterol/1.5% PEG lipid (“PEG-DMG”, i.e.,(1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with anaverage PEG molecular weight of 2000). Relative activity was determinedby measuring luciferase expression in the liver 4 hours followingadministration via tail vein injection as described in example 28. Theactivity was compared at a dose of 0.3 and 1.0 mg mRNA/kg and expressedas ng luciferase/g liver measured 4 hours after administration, asdescribed in Example 28.

TABLE 2 Lipids showing activity with mRNA Liver Luc Liver Luc @ 0.3 @1.0 Com- mg/kg mg/kg pound dose dose Structure MC2 4 ± 1 N/D

DLin- DMA 13 ± 3  67 ± 20

MC4 41 ± 10 N/D

XTC2 80 ± 28 237 ± 99 

MC3 198 ± 126 757 ± 528

319 (2% PEG) 258 ± 67  681 ± 203

137 281 ± 203 588 ± 303

The novel lipids of the invention and selected comparator lipids shownin Table 3 were formulated using the following molar ratio: 50% cationiclipid/10% distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5%PEG lipid (“PEG-DMA”2-[2-(ω-methoxy(polyethyleneglycol₂₀₀₀)ethoxy]-N,N-ditetradecylacetamide).Relative activity was determined by measuring luciferase expression inthe liver 4 hours following administration via tail vein injection asdescribed in Example 28. The activity was compared at a dose of 0.3 and1.0 mg mRNA/kg and expressed as ng luciferase/g liver measured 4 hoursafter administration, as described in Example 28. A plot of selecteddata is given in FIG. 3 (from top to bottom: circle=compound 10;triangle=compound 6; square=MC3).

TABLE 3 Exemplary Cationic lipids and Comparator Lipids Liver Luc LiverLuc @ 0.3 @ 1.0 mg/kg mg/kg (ng luc/g (ng luc/g No. pK_(a) liver) liver)Structure MC3 6.09 603 ± 150 2876 ± 622

A 7.05 * *

B 6.17 95 ± 41 1131 ± 384 

C 6.36 24 ± 4  77 ± 19

 1 5.64 54 ± 8  226 ± 20 

 5 6.27 603 ± 167 3640 ± 601 

 6 6.14 19 ± 4  211 ± 119

 7 5.93 833 ± 401 8859 ± 780 

 8 5.35 105 ± 98  1238 ± 153 

 9 6.27 2381 ± 1162 17157 ± 2470

10 6.16 2379 ± 93  26181 ± 2900

11 6.13 2273 ± 294  16502 ± 4301

12 6.21 3336 ± 1394 13577 ± 1948

13 6.22 1537 ± 777  10907 ± 2032

14 6.33 2851 ± 438  15445 ± 3693

15 6.32 2708 ± 924  15930 ± 4711

16 6.37 231 ± 100 1185 ± 838 

17 6.29 837 ± 260 6703 ± 689 

24 6.14 1120 ± 376 7425 ± 2810

35 5.97 1083 ± 350  8554 ± 4587

36 6.13 541 ± 91  4736 ± 980 

37 5.61 * *

38 6.45 905 ± 443 5353 ± 2082

39 6.45 779 ± 82  5180 ± 2116

40 6.57 753 ± 156 2203 ± 1555

41 ND^(†) 832 ± 298 7437 ± 1612

* not tested; pKa out of range ^(†)not determined

Example 30 Determination of PK_(A) of Formulated Lipids

As described elsewhere, the pKa of formulated cationic lipids iscorrelated with the effectiveness of LNPs for delivery of nucleic acids(see Jayaraman et al, Angewandte Chemie, International Edition (2012),51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176(2010)). The preferred range of pKa is ˜5 to ˜7. The pK_(a) of eachcationic lipid was determined in lipid nanoparticles using an assaybased on fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid(TNS). Lipid nanoparticles comprising of cationiclipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) in PBS at aconcentration of 0.4 mM total lipid are prepared using the in-lineprocess as described in Example 28. TNS was prepared as a 100 μM stocksolution in distilled water. Vesicles were diluted to 24 μM lipid in 2mL of buffered solutions containing, 10 mM HEPES, 10 mM MES, 10 mMammonium acetate, 130 mM NaCl, where the pH ranged from 2.5 to 11. Analiquot of the TNS solution was added to give a final concentration of 1μM and following vortex mixing fluorescence intensity was measured atroom temperature in a SLM Aminco Series 2 Luminescence Spectrophotometerusing excitation and emission wavelengths of 321 nm and 445 nm. Asigmoidal best fit analysis was applied to the fluorescence data and thepK_(a) was measured as the pH giving rise to half-maximal fluorescenceintensity (see FIG. 2).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, includingU.S. Provisional Patent Application Ser. No. 62/186,210, filed Jun. 29,2015, are incorporated herein by reference, in their entirety. Aspectsof the embodiments can be modified, if necessary to employ concepts ofthe various patents, applications and publications to provide yetfurther embodiments. These and other changes can be made to theembodiments in light of the above-detailed description. In general, inthe following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

The invention claimed is:
 1. A compound having a structure of Formula IAor IB:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: L¹ and L² are each independently —O(C═O)—, —(C═O)O—,—C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, —SC(═O)—, —NR^(a)C(═O)—,—C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—; G³is C₁-C₆ alkylene; R^(a) is H or C₁-C₁₂ alkyl; R^(1a) and R^(1b) are, ateach occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or (b)R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(1b) and thecarbon atom to which it is bound to form a carbon-carbon double bond;R^(2a) and R^(2b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(2b) and the carbon atom to which it is bound to form acarbon-carbon double bond; R^(3a) and R^(3b) are, at each occurrence,independently either (a): H or C₁-C₁₂ alkyl; or (b) R^(3a) is H orC₁-C₁₂ alkyl, and R^(3b) together with the carbon atom to which it isbound is taken together with an adjacent R^(3b) and the carbon atom towhich it is bound to form a carbon-carbon double bond; R^(4a) and R^(4b)are, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or(b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) together with the carbonatom to which it is bound is taken together with an adjacent R^(4b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond; R⁵ and R⁶ are each independently H or methyl; R⁷ is C₆-C₁₆ alkyl;R⁸ and R⁹ are each independently C₁-C₁₂ alkyl; or R⁸ and R⁹, togetherwith the nitrogen atom to which they are attached, form a 5, 6 or7-membered heterocyclic ring; a, b, c and d are each independently aninteger from 1 to 24; and x is 0, 1 or
 2. 2. The compound of claim 1,wherein: L¹ and L² are each independently —O(C═O)— or —(C═O)O—.
 3. Thecompound of claim 1, having a structure of formula (IA).
 4. The compoundof claim 1, wherein one of L¹ or L² is —O(C═O)—.
 5. The compound ofclaim 4, wherein each of L¹ and L² are —O(C═O)—.
 6. The compound ofclaim 1, wherein one of L¹ or L² is —(C═O)O—.
 7. The compound of claim6, wherein each of L¹ and L² is —(C═O)O—.
 8. The compound of claim 1,wherein for at least one occurrence of R^(1a) and R^(1b), R^(1a) is H orC₁-C₁₂ alkyl, and R^(1b) together with the carbon atom to which it isbound is taken together with an adjacent R^(1b) and the carbon atom towhich it is bound to form a carbon-carbon double bond.
 9. The compoundof claim 1, wherein for at least one occurrence of R^(4a) and R^(4b),R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(4b) and thecarbon atom to which it is bound to form a carbon-carbon double bond.10. The compound of claim 1, wherein for at least one occurrence ofR^(2a) and R^(2b), R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together withthe carbon atom to which it is bound is taken together with an adjacentR^(2b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.
 11. The compound of claim 1, wherein for at least oneoccurrence of R^(3a) and R^(3b), R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(3b) and the carbon atom to which it is bound to forma carbon-carbon double bond.
 12. The compound of claim 1, wherein a, b,c and d are each independently an integer from 2 to
 12. 13. The compoundof claim 12, wherein a, b, c and d are each independently an integerfrom 5 to
 9. 14. The compound of claim 1, wherein at least one ofR^(1a), R^(2a), R^(3a) and R^(4a) is H.
 15. The compound of claim 1,wherein R^(1a), R^(2a), R^(3a) and R^(4a) are H at each occurrence. 16.The compound of claim 1, wherein at least one of R^(1a), R^(2a), R^(3a)and R^(4a) is C₁-C₈ alkyl.
 17. The compound of claim 16, wherein C₁-C₈alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,tert-butyl, n-hexyl or n-octyl.
 18. The compound of claim 1, wherein atleast one of R^(1b), R^(2b), R^(3b) and R^(4b) is H.
 19. The compound ofclaim 1, wherein R^(1b), R^(2b), R^(3b) and R^(4b) are H at eachoccurrence.
 20. The compound of claim 1, wherein one of R⁵ or R⁶ ismethyl.
 21. The compound of claim 1, wherein each of R⁵ and R⁶ ismethyl.
 22. The compound of claim 1, wherein R⁷ is C₆-C₉ alkyl.
 23. Thecompound of claim 1, wherein R⁷ is substituted with —(C═O)OR^(b),—O(C═O)R^(b), —C(═O)R^(b), —OR^(b), —S(O)_(x)R^(b), —S—SR^(b),—C(═O)SR^(b), —SC(═O)R^(b), —NR^(a)R^(b), —NR^(a)C(═O)R^(b),—C(═O)NR^(a)R^(b), —NR^(a)C(═O)NR^(a)R^(b), —OC(═O)NR^(a)R^(b),—NR^(a)C(═O)OR^(b), —NR^(a)S(O)_(x)NR^(a)R^(b), —NR^(a)S(O)_(x)R^(b) or—S(O)_(x)NR^(a)R^(b), wherein: R^(a) is H or C₁-C₁₂ alkyl; R^(b) isC₁-C₁₅ alkyl; and x is 0, 1 or
 2. 24. The compound of claim 23, whereinR⁷ is substituted with —(C═O)OR^(b) or —O(C═O)R^(b).
 25. The compound ofclaim 23, wherein R^(b) is branched C₁-C₁₅ alkyl.
 26. The compound ofclaim 25, wherein R^(b) has one of the following structures:


27. The compound of claim 1, wherein at least one of R⁸ or R⁹ is methyl.28. The compound of claim 27, wherein each of R⁸ and R⁹ is methyl. 29.The compound of claim 1, wherein R⁸ and R⁹, together with the nitrogenatom to which they are attached, form a 5, 6 or 7-membered heterocyclicring.
 30. The compound of claim 29, wherein the heterocyclic ring ispyrrolidinyl.
 31. The compound of claim 29, wherein the heterocyclicring is piperazinyl.
 32. The compound of claim 1, wherein G³ is C₂-C₄alkylene.
 33. The compound of claim 1, wherein G³ is C₃ alkylene. 34.The compound of claim 1, having one of the following structures:


35. A composition comprising the compound of claim 1 and a therapeuticagent.
 36. The composition of claim 35, further comprising one or moreexcipient selected from neutral lipids, steroids and polymer conjugatedlipids.
 37. The composition of claim 36, wherein the compositioncomprises one or more neutral lipids selected from DSPC, DPPC, DMPC,DOPC, POPC, DOPE and SM.
 38. The composition of claim 37, wherein theneutral lipid is DSPC.
 39. The composition of claim 36, wherein thesteroid is cholesterol.
 40. The composition of claim 39, wherein themolar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.41. The composition of claim 36, wherein the polymer conjugated lipid isa pegylated lipid.
 42. The composition of claim 41, wherein the molarratio of the compound to the pegylated lipid ranges from about 100:1 toabout 25:1.
 43. The composition of claim 41, wherein the pegylated lipidis PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEGdialkyoxypropylcarbamate.
 44. The composition of claim 41, wherein thepegylated lipid has the following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R¹⁰ and R¹¹ are each independently a straight or branched,saturated or unsaturated alkyl chain containing from 10 to 30 carbonatoms, wherein the alkyl chain is optionally interrupted by one or moreester bonds; and z has a mean value ranging from 30 to
 60. 45. Thecomposition of claim 44, wherein R¹⁰ and R¹¹ are each independentlystraight, saturated alkyl chains containing from 12 to 16 carbon atoms.46. The composition of claim 44, wherein the average z is about
 45. 47.The composition of claim 35, wherein the molar ratio of the compound tothe neutral lipid ranges from about 2:1 to about 8:1.
 48. Thecomposition of claim 35, wherein the therapeutic agent comprises anucleic acid.
 49. The composition of claim 48, wherein the nucleic acidis selected from antisense and messenger RNA.
 50. A method foradministering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing the composition of claim 35 andadministering the composition to the patient.
 51. The compound of claim1, having a structure of formula (IB).